The catalase-peroxidase PiCP1 plays a critical role in abiotic stress resistance, pathogenicity and asexual structure development in Phytophthora infestans.

Catalase-peroxidase is a heme oxidoreductase widely distributed in bacteria and lower eukaryotes. In this study, we identified a catalase-peroxidase PiCP1 (PITG_05579) in Phytophthora infestans. PiCP1 had catalase/peroxidase and secretion activities and was highly expressed in sporangia and upregulated in response to oxidative and heat stresses. Compared with wild type, PiCP1-silenced transformants (STs) had decreased catalase activity, reduced oxidant stress resistance and damped cell wall integrity. In contrast, PiCP1-overexpression transformants (OTs) demonstrated increased tolerance to abiotic stresses and induced the upregulation of PR genes in the host salicylic acid pathway. The high concentration of PiCP1 can also induced callose deposition in plant tissue. Importantly, both STs and OTs have severely reduced sporangia formation and zoospore releasing rate, but the sporangia germination rate and type varied depending on environmental conditions. Comparative sequence analyses show that catalase-peroxidases are broadly distributed and highly conserved among soil-borne plant parasitic oomycetes, but not in freshwater-inhabiting or strictly plants-inhabiting oomycetes. In addition, we found that silencing PiCP1 downregulated the expression of PiCAT2. These results revealed the important roles of PiCP1 in abiotic stress resistance, pathogenicity and in regulating asexual structure development in response to environmental change. Our findings provide new insights into catalase-peroxidase functions in eukaryotic pathogens.

LysM protein BdLM1 of Botryosphaeria dothidea plays an important role in full virulence and inhibits plant immunity by binding chitin and protecting hyphae from hydrolysis.

Botryosphaeria dothidea infects hundreds of woody plants and causes a severe economic loss to apple production. In this study, we characterized BdLM1, a protein from B. dothidea that contains one LysM domain. BdLM1 expression was dramatically induced at 6 h post-inoculation in wounded apple fruit, strongly increased at 7 d post-inoculation (dpi), and peaked at 20 dpi in intact shoots. The knockout mutants of BdLM1 had significantly reduced virulence on intact apple shoots (20%), wounded apple shoots (40%), and wounded apple fruit (40%). BdLM1 suppressed programmed cell death caused by the mouse protein BAX through Agrobacterium-mediated transient expression in Nicotiana benthamiana, reduced H2O2 accumulation and callose deposition, downregulated resistance gene expression, and promoted Phytophthora nicotianae infection in N. benthamiana. Moreover, BdLM1 inhibited the active oxygen burst induced by chitin and flg22, bound chitin, and protected fungal hyphae against degradation by hydrolytic enzymes. Taken together, our results indicate that BdLM1 is an essential LysM effector required for the full virulence of B. dothidea and that it inhibits plant immunity. Moreover, BdLM1 could inhibit chitin-triggered plant immunity through a dual role, i.e., binding chitin and protecting fungal hyphae against chitinase hydrolysis.

Red cell distribution width is associated with stroke severity and unfavorable functional outcomes in ischemic stroke.

Background: Red blood cell distribution width (RDW) is considered to be related to coronary heart disease and heart failure and all-cause mortality, but its relationship with acute ischemic stroke is still unclear. In this study, we aimed to explore the relationship between RDW and the stroke severity and functional outcomes of ischemic stroke. Methods: We retrospectively reviewed patients with acute ischemic stroke between September 2016 and January 2020. Demographic, clinical, stroke complications, laboratory data, and treatment were collected for all patients. Stroke severity and functional outcomes were evaluated by NIHSS score, modified Rankin Scale (mRS), and Barthel Index (BI) at 3 months. Furthermore, multiple logistic regression analysis was used to assess the relationship between RDW and stroke severity and functional outcomes. Results: A total of 629 patients with acute ischemic stroke were included and were categorized into four groups according to the quartiles of RDW (< 12.4, 12.4-12.9, 13.0-13.4, > 13.4). After multivariable analysis, higher RDW was directly associated with moderate to severe stroke (OR 2.21, 95% CI, 1.30-3.75, P = 0.003), mRS score of 3-6 at 3 months (OR 1.86, 95% CI, 1.02-3.41, P = 0.044), and BI score below 85 at 3 months (OR 2.27, 95% CI, 1.25-4.12, P = 0.007) in patients with ischemic stroke. Conclusion: Our results demonstrate that RDW is associated with stroke severity and unfavorable functional outcomes at 3 months in patients with ischemic stroke.

Nomogram to predict 3-month unfavorable outcome after thrombectomy for stroke.

BACKGROUND: Mechanical thrombectomy (MT) is an effective treatment for large-vessel occlusion in acute ischemic stroke, however, only some revascularized patients have a good prognosis. For stroke patients undergoing MT, predicting the risk of unfavorable outcomes and adjusting the treatment strategies accordingly can greatly improve prognosis. Therefore, we aimed to develop and validate a nomogram that can predict 3-month unfavorable outcomes for individual stroke patient treated with MT. METHODS: We analyzed 258 patients with acute ischemic stroke who underwent MT from January 2018 to February 2021. The primary outcome was a 3-month unfavorable outcome, assessed using the modified Rankin Scale (mRS), 3-6. A nomogram was generated based on a multivariable logistic model. We used the area under the receiver-operating characteristic curve to evaluate the discriminative performance and used the calibration curve and Spiegelhalter's Z-test to assess the calibration performance of the risk prediction model. RESULTS: In our visual nomogram, gender (odds ratio [OR], 3.40; 95%CI, 1.54-7.54), collateral circulation (OR, 0.46; 95%CI, 0.28-0.76), postoperative mTICI (OR, 0.06; 95%CI, 0.01-0.50), stroke-associated pneumonia (OR, 5.76; 95%CI, 2.79-11.87), preoperative Na (OR, 0.82; 95%CI, 0.72-0.92) and creatinine (OR, 1.02; 95%CI, 1.01-1.03) remained independent predictors of 3-month unfavorable outcomes in stroke patients treated with MT. The area under the nomogram curve was 0.8791 with good calibration performance (P = 0.873 for the Spiegelhalter's Z-test). CONCLUSIONS: A novel nomogram consisting of gender, collateral circulation, postoperative mTICI, stroke-associated pneumonia, preoperative Na and creatinine can predict the 3-month unfavorable outcomes in stroke patients treated with MT.

The Cutinase Bdo_10846 Play an Important Role in the Virulence of Botryosphaeria dothidea and in Inducing the Wart Symptom on Apple Plant.

Botryosphaeria dothidea is a pathogen with worldwide distribution, infecting hundreds of species of economically important woody plants. It infects and causes various symptoms on apple plants, including wart and canker on branches, twigs, and stems. However, the mechanism of warts formation is unclear. In this study, we investigated the mechanism of wart formation by observing the transection ultrastructure of the inoculated cortical tissues at various time points of the infection process and detecting the expression of genes related to the pathogen pathogenicity and plant defense response. Results revealed that wart induced by B. dothidea consisted of proliferous of phelloderm cells, the newly formed secondary phellem, and the suberized phelloderm cells surrounding the invading mycelia. The qRT-PCR analysis revealed the significant upregulation of apple pathogenesis-related and suberification-related genes and a pathogen cutinase gene Bdo_10846. The Bdo_10846 knockout transformants showed reduced cutinase activity and decreased virulence. Transient expression of Bdo_10846 in Nicotiana benthamiana induced ROS burst, callose formation, the resistance of N. benthamiana to Botrytis cinerea, and significant upregulation of the plant pathogenesis-related and suberification-related genes. Additionally, the enzyme activity is essential for the induction. Virus-induced gene silencing demonstrated that the NbBAK1 and NbSOBIR1 expression were required for the Bdo_10846 induced defense response in N. benthamiana. These results revealed the mechanism of wart formation induced by B. dothidea invasion and the important roles of the cutinase Bdo_10846 in pathogen virulence and in inducing plant immunity.

Candidate Effectors from Botryosphaeria dothidea Suppress Plant Immunity and Contribute to Virulence.

Fungal effectors play important roles in host-pathogen interactions. Botryosphaeria dothidea is an ascomycetous fungus that is responsible for the diseases of hundreds of woody plant species, including apple ring rot, which seriously affects apples worldwide. However, little is known about the effectors of B. dothidea. In this study, we analyzed the B. dothidea genome and predicted 320 candidate effector genes, 124 of which were successfully amplified and cloned. We investigated the effects of these genes on plant cell death in Nicotiana benthamiana while using a transient expression system. Twenty-four hours after initial inoculation with Agrobacterium tumefaciens cells carrying candidate effectors, the infiltrated leaves were challenged with A. tumefaciens cells carrying the BAX gene. In total, 116 candidate effectors completely inhibited, while one partially inhibited, the programmed cell death (PCD) of N. benthamiana induced by BAX, whereas seven candidate effectors had no effect. We then further tested seven candidate effectors able to suppress BAX-triggered PCD (BT-PCD) and found that they all completely inhibited PCD triggered by the elicitors INF1, MKK1, and NPK1. This result suggests that these effectors were activated in order to suppress pathogen-associated molecular pattern-triggered immunity. The signal peptides of these candidate effectors exhibited secretory activity in yeast (pSUC2 vector). Moreover, the respective deletion of Bdo_11198 and Bdo_12090 significantly reduced the virulence of B. dothidea. These results suggest that these effectors play important roles in the interaction of B. dothidea with its hosts.

Left Atrial Enlargement is Associated with Stroke Severity with Cardioembolic and Cryptogenic Subtypes in a Chinese Population.

BACKGROUND: Left atrial enlargement is associated with increased risk for stroke. However, few studies that evaluated the correlation between left atrial size and ischemic stroke severity. In this study, we aim to evaluate the association between left atrial size and stroke severity, especially with cardioembolic and cryptogenic stroke in the Chinese population. METHODS: A total of 1271 patients with acute ischemic stroke were included in this study. Echocardiographic left atrial diameter was measured and indexed to height. Stroke severity was assessed at admission with the National Institutes of Health Stroke Scale (NIHSS). Moderate-to-severe neurologic deficit was defined as NIHSS greater than or equal to 5. Patients were divided into mild, moderate, or severe abnormal left atrial size by tertile distribution. Binary logistic regression analysis was used to identify independent predictors of severe stroke after adjustment. RESULTS: Among all enrolled patients, 328 (25.8%) were classified into moderate-to severe stroke severity (NIHSS ≥ 5). In the multivariable model, compared with the lowest tertile of left atrial size, the odds ratio for moderate-to-severe neurologic deficit was 0.902 (95% CI, 0.644-1.264, P = .550) when left atrial size was the highest tertile. Of all patients, 190 patients were further categorized as cardioembolic and cryptogenic subtypes, and 70 (36.8%) were classified into moderate-to-severe stroke severity. After adjusting for confounders, compared with the lowest tertile, the top tertile of left atrial size was significantly associated with moderate-to-severe stroke (3.156, 95% CI, 1.143-8.711, P = .027). CONCLUSION: Left atrial enlargement was associated with more severe initial neurologic deficits of embolic subtypes (cardioembolic and cryptogenic stroke) in patients with acute ischemic stroke.

A proper PiCAT2 level is critical for sporulation, sporangium function, and pathogenicity of Phytophthora infestans.

Catalase is present in prokaryotic and eukaryotic organisms and is important for the protective effects of the antioxidant system against free radicals. Many studies have confirmed that catalase is required for the growth, development, and pathogenesis of bacteria, plants, animals, and fungi. However, there has been relatively little research on the catalases in oomycetes, which form an important group of fungus-like eukaryotes that produce zoosporangia. In this study, we detected two Phytophthora infestans genes encoding catalases, but only PiCAT2 exhibited catalase activity in the sporulation stage and was highly produced during asexual reproduction and in the late infection stage. Compared with the wild-type strain, the PiCAT2-silenced P. infestans transformants were more sensitive to abiotic stress, were less pathogenic, and had a lower colony expansion rate and lower PiMPK7, PiVPS1, and PiGPG1 expression levels. In contrast, the PiCAT2-overexpressed transformants were slightly less sensitive to abiotic stress. Interestingly, increasing and decreasing PiCAT2 expression from the normal level inhibited sporulation, germination, and infectivity, and down-regulated PiCdc14 expression, but up-regulated PiSDA1 expression. These results suggest that PiCAT2 is required for P. infestans mycelial growth, asexual reproduction, abiotic stress tolerance, and pathogenicity. However, a proper PiCAT2 level is critical for the formation and normal function of sporangia. Furthermore, PiCAT2 affects P. infestans sporangial formation and function, pathogenicity, and abiotic stress tolerance by regulating the expression of cell cycle-related genes (PiCdc14 and PiSDA1) and MAPK pathway genes. Our findings provide new insights into catalase functions in eukaryotic pathogens.

Personalised antiplatelet therapy based on pharmacogenomics in acute ischaemic minor stroke and transient ischaemic attack: study protocol for a randomised controlled trial.

INTRODUCTION: Antiplatelet therapy combining aspirin and clopidogrel is considered to be a key intervention for acute ischaemic minor stroke (AIMS) and transient ischaemic attack (TIA). However, the interindividual variability in response to clopidogrel resulting from the polymorphisms in clopidogrel metabolism-related genes has greatly limited its efficacy. To date, there are no reports on individualised antiplatelet therapy for AIMS and TIA based on the genetic testing and clinical features. Therefore, we conduct this randomised controlled trial to validate the hypothesis that the individualised antiplatelet therapy selected on the basis of a combination of genetic information and clinical features would lead to better clinical outcomes compared with the standard care based only on clinical features in patients with AIMS or TIA. METHODS AND ANALYSIS: This trial will recruit 2382 patients with AIMS or TIA who meet eligibility criteria. Patients are randomly assigned in a 1:1 ratio to pharmacogenetic group and standard group. Both groups receive a loading dose of 300 mg aspirin and 300 mg clopidogrel on day 1, followed by 100 mg aspirin per day on days 2-365. The P2Y12 receptor antagonist is selected by the clinician according to the genetic information and clinical features for pharmacogenetic group and clinical features for the standard group on days 2-21. The primary efficacy endpoint is a new stroke event (ischaemic or haemorrhagic) that happens within 1 year. The secondary efficacy endpoint is analysed as the individual or composite outcomes of the new clinical vascular event (ischaemic stroke, haemorrhagic stroke, myocardial infarction or vascular death). Baseline characteristics and outcomes after treatment will be evaluated. ETHICS AND DISSEMINATION: This protocol has been approved by the ethics committee of Yangpu Hospital, Tongji University School of Medicine (No. LL-2018-KY-012). We will submit the results of this trial for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ChiCTR1800019911; Pre-results.

Clinical characteristics and risk score for poor clinical outcome of acute ischemic stroke patients treated with intravenous thrombolysis therapy.

BACKGROUND: Tissue plasminogen activator (t-PA) is an effective therapy for acute ischemic stroke, but some patients still have poor clinical outcome. In this study, we investigated clinical characteristics of stroke patients and determined predictors for poor clinical outcome in response to t-PA treatment. METHODS: Clinical data from 247 patients were retrospectively reviewed. Clinical parameters that were associated with survival of patients were analyzed. Areas under receiver operating characteristic curves (ROC) were used to determine the feasibility of using various combinations of the clinical parameters to predict poor clinical response. The clinical outcome was defined according to the changes in Modified Rankin Scale. RESULTS: Overall, 145 patients had improved/complete recovery, 73 had no change, and 29 had worsening conditions or died during the in-clinic period. A univariate analysis showed that baseline characteristics including age, CRP, blood glucose level, systolic blood pressure, and admission NIHSS were significantly different (p < 0.05) among patients with different clinical outcome. A further multivariate analysis was then performed. Variables associated with poor clinical outcome (worsening/death) (p < 0.1) were included in the logistic regression model. Four parameters were retained in the model: Age, CRP, Blood glucose level, and Systolic blood pressure (ACBS). To allow a convenient usage of the ACBS classifier, the parameters were put into a scoring system, and the score at 7.7 was chosen as a cut-off. The ROC curve of this ACBS classifier has an area under the curve (AUC) of 0.7788, higher than other individual parameters. The ACBS classifier provided enhanced sensitivity of 69.2% and specificity of 74.3%. CONCLUSION: The ACBS classifier provided a satisfactory power in estimating the patients' clinical outcome. After further validating, the classifier may provide important information to clinicians for making clinical decisions.

Detection and Identification of Six Monilinia spp. Causing Brown Rot Using TaqMan Real-Time PCR from Pure Cultures and Infected Apple Fruit.

Brown rot is a severe disease affecting stone and pome fruit. This disease was recently confirmed to be caused by the following six closely related species: Monilinia fructicola, M. laxa, M. fructigena, Monilia polystroma, M. mumecola, and M. yunnanensis. Because of differences in geographic distributions, some of these species are important quarantine pathogens in certain countries. In this study, we developed TaqMan real-time polymerase chain reaction (PCR) assays to detect and identify the six species. Primer pairs and probes were designed for Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma based on sequence differences in the laccase-2 genes. Additionally, based on sequence differences in the elongation factor genes, primer pairs and probes were designed for Monilia mumecola and M. yunnanensis. The real-time PCR assays were able to specifically identify the target pathogens, with detection limits of 10 to 100 fg of DNA, which is equivalent to one to seven conidia. The assays were also able to detect the target pathogens in a mixed DNA sample comprising all six Monilinia spp. and related species. The real-time PCR assays accurately detected target fungi from infected apple fruit. Furthermore, the identification results were consistent with those of traditional morphological methods.

Monilinia Species Associated with Brown Rot of Cultivated Apple and Pear Fruit in China.

Monilinia isolates were collected from major apple and pear production regions in China from 2004 to 2011 and identified based on their morphological characteristics and three highly conserved loci. The 247 isolates belonged to three species: Monilinia fructicola, Monilia yunnanensis, and Monilia polystroma. M. yunnanensis was the most prevalent (77%), followed by M. polystroma (20%) and Monilinia fructicola (3%). Monilia yunnanensis is primarily distributed in the south, north, and west of China; M. polystroma is limited to the north and east; and Monilinia fructicola was detected only from a few samples from the north and east. Phylogenetic analysis based on internal transcribed spacer, ß-tubulin, and laccase (lcc2) genes suggested that Monilia yunnanensis, M. polystroma, and Monilinia fructigena are closely related, and Monilia yunnanensis is more distantly related. We also found that these three species do not show consistent differences in morphological characteristics, including colony morphology, colony expansion rate, conidial characteristics, and the amount of stroma produced in culture. Thus, these three species are more like phylogenetic species in the process of speciation. In addition, a set of species-specific primers based on single-nucleotide polymorphisms and deletions in the lcc2 gene region were designed and a conventional polymerase chain reaction method successfully developed for differentiating Monilinia fructicola, Monilia yunnanensis, M. polystroma, and Monilinia laxa from the other species.

Phytophthora infestans field isolates from Gansu province, China are genetically highly diverse and show a high frequency of self fertility.

The genetic diversity of 85 isolates of Phytophthora infestans collected in 2007 from Gansu province in China was determined and compared with 21 isolates collected before 2004. Among them, 70 belonged to the A1 mating type and 15 were self-fertile (SF). The mitochondrial DNA haplotypes revealed both Ia (25%) and IIa (75%) haplotypes. Metalaxyl resistance occurred with high frequency (54%) in Gansu. Simple sequence repeat (SSR) genotyping revealed 26 genotypes (13 from the Tianshui region) among the 85 isolates, and 18 genotypes among the 21 isolates collected before 2004, without overlap in genotypes detected in the two groups. Cluster analysis showed clear subdivisions within the different mating type isolates. Among Gansu's isolates, Nei's and Shannon's diversity indices were highest in isolates collected in Tianshui where both A1 and SF isolates were found. Analysis of molecular variance of isolates from Gansu indicated that 51% and 49% of the variance was explained by within-area and among-area variance, respectively. The results suggest that the occurrence of SF isolates increases the risk of sexual reproduction, the formation of oospore as initial inocula in the field, and affects the genotypic diversity in the population.

Population Structure of Brown Rot Fungi on Stone Fruits in China.

In total, 455 Monilinia isolates from stone fruits collected from several provinces (cities) in China from 2003 to 2009 were identified to species based on morphological characteristics, molecular identification, and the sequence of the internal transcribed spacer (ITS) regions 1 and 2 and the 5.8S gene of the ribosomal RNA. Overall, four species were detected (Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma). M. fructicola was the most prevalent (93.0%) followed by M. fructigena (4.8%), M. laxa (2.0%), and Monilia polystroma (0.2%). M. fructicola and M. fructigena were found on peach, plum, and apricot; M. laxa was found only on apricot, cherry (in an organic orchard), and wild peach; and Monilia polystroma was found only on plum in Heilongjiang. The pathogenicity of Monilinia fructicola, M. laxa, and M. fructigena did not significantly differ on wounded nectarine and apricot, indicating that the differences in frequency of occurrence were not linked to virulence. Phylogenetic analysis based on ITS sequences showed that the isolates of M. laxa and M. fructigena from China differed from isolates of these species from other countries, and that the difference led to the separation of the isolates from China and those from other countries into different phylogenetic groups. Further study is needed to determine whether they are cryptic species.

Genetic structure of Phytophthora infestans populations in China indicates multiple migration events.

One hundred isolates of Phytophthora infestans collected from 10 provinces in China between 1998 and 2004 were analyzed for mating type, metalaxyl resistance, mitochondrial DNA (mtDNA) haplotype, allozyme genotype, and restriction fragment length polymorphism (RFLP) with the RG-57 probe. In addition, herbarium samples collected in China, Russia, Australia, and other Asian countries were also typed for mtDNA haplotype. The Ia haplotype was found during the first outbreaks of the disease in China (1938 and 1940), Japan (1901, 1930, and 1931), India (1913), Peninsular Malaysia (1950), Nepal (1954), The Philippines (1910), Australia (1917), Russia (1917), and Latvia (1935). In contrast, the Ib haplotype was found after 1950 in China on both potato and tomato (1952, 1954, 1956, and 1982) and in India (1968 and 1974). Another migration of a genotype found in Siberia called SIB-1 (Glucose-6-phosphate isomerase [Gpi] 100/100, Peptidase [Pep] 100/100, IIa mtDNA haplotype) was identified using RFLP fingerprints among 72% of the isolates and was widely distributed in the north and south of China and has also been reported in Japan. A new genotype named CN-11 (Gpi 100/111, Pep 100/100, IIb mtDNA haplotype), found only in the south of China, and two additional genotypes (Gpi 100/100, Pep 100/100, Ia mtDNA haplotype) named CN-9 and CN-10 were identified. There were more diverse genotypes among isolates from Yunnan province than elsewhere. The SIB-1 (IIa) genotype is identical to those from Siberia, suggesting later migration of this genotype from either Russia or Japan into China. The widespread predominance of SIB-1 suggests that this genotype has enhanced fitness compared with other genotypes found. Movement of the pathogen into China via infected seed from several sources most likely accounts for the distribution of pathogen genotypes observed. MtDNA haplotype evidence and RFLP data suggest multiple migrations of the pathogen into China after the initial introduction of the Ia haplotype in the 1930s.